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Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR and SR-related proteins. These proteins are characterized by domains rich in arginine and serine (RS domains), which are essential to alternative splicing and in vivo phase separation. However, they are also responsible for a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a co-solute. We find that this RS-mimic peptide forms interactions similar to those of the protein’s RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1’s RNA Recognition Motifs (RRMs) through electrostatic and cation-pi interactions. Analysis of RRM domains from human SR proteins indicates that these sites are conserved across the protein family. In addition to opening an avenue to previously unavailable proteins, our work provides insight into how SR proteins phase separate and participate in nuclear speckles. 

Abstract Serine/arginine‐rich splicing factor 1 (SRSF1) is key in the mRNA lifecycle including transcription, splicing, nonsense‐mediated decay, and nuclear export. Consequently, its dysfunction is linked to cancers, viral evasion, and developmental disorders. The functionality of SRSF1 relies on its interactions with other proteins and RNA molecules. These processes are regulated by phosphorylation of its unstructured arginine/serine‐rich tail (RS). Here, we characterize how phosphorylation affects SRSF1's protein and RNA interaction and phase separation. Using NMR paramagnetic relaxation enhancement and chemical shift perturbation, we find that when unphosphorylated, SRSF1's RS interacts with its first RNA‐recognition motif (RRM1). Phosphorylation of RS decreases its interactions with the protein‐binding site of RRM1 and increases its interactions with the RNA‐binding site of RRM1. This change in SRSF1's intramolecular interactions increases the availability of protein‐interacting sites on RRM1 and weakens RNA binding of SRSF1. Phosphorylation alters the phase separation of SRSF1 by diminishing the role of arginine in intermolecular interactions. These findings provide an unprecedented view of how SRSF1 influences the early‐stage spliceosome assembly.